De-regulation of the C-MYC oncogene is among the most frequent molecular abnormalities in human cancer. c-Myc is a bHLH-ZIP transcription factor whose dimerization with another bHLH-ZIP protein, Max, is necessary for transformation and all other biological activities. That c-Myc is indispensable for cell proliferation, that most normal cells are non-cycling and thus non-c-Myc-expressing, and that transient inhibition of c-Myc may be sufficient to achieve permanent tumor regression, makes therapeutic targeting of the oncoprotein highly appealing. We have previously identified 7 low molecular weight compounds that abrogate/prevent bHLH-ZIP-mediated c-Myc-Max heterodimerization (Myc compounds). Non structure- guided (i.e. unbiased) modifications of one of these has resulted in the generation of 2nd generation analogs with up to 8-fold enhanced potencies, thus demonstrating that significant improvements in compound efficacy can be obtained even in the absence of a model of the compound in association with its target site. In order to identify even better analogs in a more rational and meaningful way, we have demonstrated that 3 of our original Myc compounds bind to distinct sites on the intrinsically disordered c- Myc bHLH-ZIP monomer, alter its secondary structure, and prevent its association with Max. The remaining 4 compounds are redundant and bind to one of these sites. Using NMR spectroscopy, we have generated structural models of the first three compounds in association with synthetic peptides comprising their cognate binding sites. Thus, in Specific Aim 1, we will utilize computational methods and high throughput in silico screens to identify new and more potent 3rd generation analogs whose structures are rationally predicated on those of the original 3 founding members. In Specific Aim 2, we will compare the abilities of these new compounds to inhibit c-Myc-Max association and c-Myc-mediated neoplastic growth. In Specific Aim 3, we will conduct in vivo pharmacologic testing of the most promising analogs prioritized by their ADMET profiles. Finally, in Specific Aim 4, selected analogs identified in Specific Aims 2 and 3 will be chemically linked in order to generate 4th generation compounds capable of simultaneous binding to two distinct sites on the c-Myc bHLH-ZIP domain. This proposal is highly interdisciplinary and interactive in that it utilizes the expertise of a pediatric oncologist/molecular biologist (Prochownik), an organic chemist (Metallo), computational biologists (Behar and Mustata), and pharmacologists (Lazo, Eiseman and Egorin). Together, this diverse, complementary, and synergistic group of investigators, from two institutions, proposes studies that promise new insights regarding the rational design of Myc compounds and the mechanisms by which they disable this critical oncoprotein.